RESUMO
Sublethal periods of hypoxia or ischemia can induce adaptive mechanisms to protect against subsequent lethal ischemic insults in a process known as ischemic preconditioning. In the present study, we developed a murine model of cerebral preconditioning using several common strains of adult mice. Animals were exposed to sublethal hypoxia (11% oxygen for 2 h) 48 h prior to a 90 min period of transient focal middle cerebral artery occlusion, induced by an intraluminal filament; injury was assessed 24 h later by TTC staining. Infarct volume in hypoxia-preconditioned animals was reduced 46%, 58%, and 64% in C57Bl/6, 129SvEv, and Swiss-Webster ND4 mice relative to their respective untreated controls. This non-invasive murine model of ischemic tolerance should be useful for elucidating the molecular basis of this protection using transgenic and knockout mice.
Assuntos
Isquemia Encefálica/fisiopatologia , Encéfalo/fisiopatologia , Precondicionamento Isquêmico , Traumatismo por Reperfusão/fisiopatologia , Animais , Infarto Cerebral/patologia , Hipóxia/fisiopatologia , Precondicionamento Isquêmico/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos EndogâmicosRESUMO
BACKGROUND AND PURPOSE: Genetically engineered mice are used to study the role of single genes in cerebral ischemia, but inherent, strain-dependent differences in neuronal vulnerability may affect experimental end points. To examine this possibility, tissue injury resulting from focal ischemia and its relationship to cerebral hemodynamics were determined in 3 common mutant mouse strains. METHODS: Permanent middle cerebral artery ligation was performed in male C57BL/6J, Balb/C, and 129X1/SvJ mice. Mean arterial blood pressure, blood gases, basal and postischemic cortical blood flow ([(14)C]iodoantipyrine autoradiography and laser-Doppler flowmetry), posterior communicating artery patency, and infarct size were determined. RESULTS: Basal cortical blood flow did not differ among strains. Ten minutes after middle cerebral artery ligation, relative red cell flow in the ischemic cortex was 6% to 7% of preischemic flow in every strain. Despite similar hemodynamics, cortical infarcts in Balb/C mice were 3-fold larger than those in 129X1/SvJ and C57BL/6J mice; infarct size in the latter 2 strains was not significantly different. The posterior communicating artery was either poorly developed or absent in >90% of the Balb/C and C57BL/6J but in <50% of the 129X1/SvJ mice. CONCLUSIONS: The extent of ischemic injury differed markedly between the 3 strains. The presence and patency of posterior communicating arteries, although variable among strains, did not affect preischemic or postischemic cortical blood flow or bear any relationship to ischemic injury. Therefore, intrinsic factors, other than hemodynamic variability, may contribute to the differences in ischemic vulnerability among strains. These findings underscore the importance of selecting genetically matched wild-type controls.
Assuntos
Antipirina/análogos & derivados , Isquemia Encefálica/genética , Modelos Animais de Doenças , Predisposição Genética para Doença/genética , Camundongos Mutantes/genética , Animais , Autorradiografia , Gasometria , Carbono , Radioisótopos de Carbono , Córtex Cerebral/irrigação sanguínea , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fluxo Sanguíneo Regional , Projetos de Pesquisa/normas , Especificidade da EspécieRESUMO
Hypoxic-ischemic (H-I) brain injury in the human perinatal period often leads to significant long-term neurobehavioral dysfunction in the cognitive and sensory-motor domains. Using a neonatal H-I injury model (unilateral carotid ligation followed by hypoxia) in postnatal day seven rats, previous studies have shown that neurotrophins, such as brain-derived neurotrophic factor (BDNF), can be protective against neural tissue loss. The present study explored potential relationships between neural protective and behavioral protective strategies in this neonatal H-I model by determining if neonatal H-I was associated with behavioral spatial learning and memory deficits and whether the neurotrophin BDNF was protective against both brain injury and spatial learning/memory dysfunction. Postnatal day seven rats received vehicle or BDNF pretreatments (intracerebroventricular injections) followed by H-I or sham treatments and then tested for spatial learning and memory on the simple place task in the Morris water maze from postnatal days 20 to 30, and their brains were histologically analyzed at 4 weeks following treatments. H-I rats with vehicle pretreatment displayed significant tissue loss in the hippocampus (including CA1 neurons), cortex, and striatum, as well as severe spatial memory deficits (e.g., short probe times). BDNF pretreatment resulted in significant protection against both H-I-induced brain tissue losses and spatial memory impairments. These findings indicate that unilateral H-I brain injury in a neonatal rodent model is associated with cognitive deficits, and that BDNF pretreatment is protective against both brain injury and spatial memory impairment.
Assuntos
Animais Recém-Nascidos/fisiologia , Asfixia Neonatal/tratamento farmacológico , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Transtornos da Memória/tratamento farmacológico , Animais , Asfixia Neonatal/patologia , Asfixia Neonatal/fisiopatologia , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Encéfalo/fisiopatologia , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/patologia , Lesões Encefálicas/fisiopatologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Contagem de Células , Modelos Animais de Doenças , Feminino , Humanos , Hipóxia-Isquemia Encefálica/patologia , Hipóxia-Isquemia Encefálica/fisiopatologia , Recém-Nascido , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/patologia , Transtornos da Memória/fisiopatologia , Degeneração Neural/tratamento farmacológico , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Gravidez , Ratos , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/fisiologiaRESUMO
Hypoxia-inducible factor-1 (HIF-1) is a heterodimer composed of HIF-1alpha and HIF-1beta protein subunits. This transcription factor is essential for the activation of hypoxia-inducible genes like erythropoietin, some glucose transporters, the glycolytic enzymes, and vascular endothelial growth factor. Because HIF-1 activation may promote cell survival in hypoxic tissues, we studied the effect of hypoxic preconditioning on HIF-1 expression in neonatal rat brain. Hypoxic preconditioning (8% O2 for 3 hours), a treatment known to protect the newborn rat brain against hypoxic-ischemic injury, markedly increased HIF-1alpha and HIF-1beta expression. To support the role of HIF-1 in protective preconditioning, we also studied the effect of two other known HIF-1 inducers, cobalt chloride (CoCl2) and desferrioxamine (DFX), on HIF-1 expression and neuroprotection in newborn brain. HIF-1alpha and HIF-1beta protein levels were markedly increased after intraperitoneal injection of CoCl2 (60 mg/kg) and moderately increased after intraperitoneal injection of DFX (200 mg/kg) 1 to 3 hours after the injections. Preconditioning with CoCl2 or DFX 24 hours before hypoxia-ischemia afforded 75 and 56% brain protection, respectively, compared with that in vehicle-injected littermate controls. Thus, HIF-1 activation could contribute to protective brain preconditioning, which could be used in high-risk deliveries and other clinical situations.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Hipóxia-Isquemia Encefálica/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição , Animais , Western Blotting , Feminino , Regulação da Expressão Gênica , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND AND PURPOSE: Hydroxyethyl starch (HES) has beneficial effects on ischemic brain injury; however, its mechanism of action remains unclear. The present study was undertaken to test the hypothesis that HES can attenuate increases in leukocyte adherence and vascular permeability in the cerebral vasculature after global cerebral ischemia induced by asphyxia. METHODS: Pial venular leukocyte adherence and permeability to sodium fluorescein were quantified in anesthetized newborn piglets by in situ fluorescence videomicroscopy through closed cranial windows during basal conditions and during 2 hours of reperfusion after global ischemia induced by 9 minutes of asphyxia. Experimental animals received HES after the asphyxial insult (10% HES 257/0.47, 600 mg/kg IV bolus 5 minutes after asphyxia, followed by 600 mg/kg per hour IV drip during reperfusion; n=9). RESULTS: A progressive and significant (P:<0.05) increase in adherent leukocytes was observed during the initial 2 hours of reperfusion after asphyxia compared with nonasphyxial controls. In this model, vascular injury, as determined by significant (P:<0.05) increases in fluorescein permeability at 2 hours of reperfusion, is largely dependent on adherent leukocytes. HES significantly reduced (P:<0.05) leukocyte adherence at 1 hour and 2 hours of reperfusion and reduced fluorescein permeability at 2 hours. HES did not change hematocrit or alter pial arteriolar diameter. CONCLUSIONS: These findings indicate that a vascular anti-inflammatory action may underlie the beneficial effects of HES in global cerebral ischemia secondary to asphyxia. Since this compound is well tolerated by patients, future preclinical and clinical studies may reveal improvements in functional outcome with the early introduction of this or similar agents after perinatal asphyxia or global ischemia.
Assuntos
Isquemia Encefálica/fisiopatologia , Circulação Cerebrovascular/efeitos dos fármacos , Derivados de Hidroxietil Amido/farmacologia , Substitutos do Plasma/farmacologia , Animais , Animais Recém-Nascidos , Anti-Inflamatórios não Esteroides/farmacologia , Asfixia , Isquemia Encefálica/etiologia , Permeabilidade Capilar/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Circulação Cerebrovascular/fisiologia , Modelos Animais de Doenças , Fluoresceína , Leucócitos/efeitos dos fármacos , Reperfusão , SuínosRESUMO
Hypoxic-ischemic (H-I) injury to the brain in the perinatal period often leads to significant long-term neurological deficits. In a model of neonatal H-I injury in postnatal day 7 rats, our previous data have shown that cell death with features of apoptosis is prominent between 6 and 24 h after H-I and that neurotrophins, particularly BDNF, can markedly protect against tissue loss. During brain development, caspase-3 is required for normal levels of programmed cell death. Utilizing an antibody specific for the activated form of caspase-3, CM1, we now show that caspase-3 is specifically activated in neuronal cell bodies and their processes beginning at 6 h and peaking 24 h following unilateral carotid ligation and exposure to hypoxia in postnatal day 7 rats. Caspase-3 activation began to occur in cortex at 6 h and in striatum and hippocampus at 12-18 h. Caspase-3 activation was also observed in developing oligodendrocytes. Intracerebroventricular injection of BDNF prior to H-I injury almost completely abolished evidence of H-I-induced caspase-3 activation in vivo. Utilizing a specific molecular marker of an apoptotic pathway, these findings demonstrate that H-I injury to the developing brain is a strong apoptotic stimulus leading to caspase-3 activation, that BDNF can block this process in vivo, and that the ability of BDNF to inhibit caspase activation and subsequent apoptosis likely accounts in large part for its protection against neuronal injury in this model.
Assuntos
Isquemia Encefálica/enzimologia , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Caspases/metabolismo , Hipóxia Encefálica/enzimologia , Animais , Animais Recém-Nascidos , Especificidade de Anticorpos , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Isquemia Encefálica/patologia , Caspase 3 , Ativação Enzimática/efeitos dos fármacos , Lateralidade Funcional , Hipóxia Encefálica/patologia , Neurônios/patologia , Neurônios/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Adenosine, an intercellular messenger that is a product of the metabolism of ATP, plays a major role in neuronal and vascular responses of the retina to alterations in oxygen delivery. Significant changes in adenosine concentration have been measured in the retina during both ischemia and during the subsequent reperfusion period which result in important, but complex, functional effects. Adenosine A1 receptor stimulation produces a protective effect during ischemia, whereas overstimulation of the A2a receptor has deleterious effects. The mechanisms underlying these findings have not been completely determined, but most likely are the result of alterations in excitotoxicity, gene expression, and blood flow. Paradoxically, prolonged increases in adenosine concentration may be injurious to the retina, a consequence of superoxide radical formation secondary to adenosine catabolism. Adenosine is a critical mediator of blood flow changes in response to ischemia. It is a significant component of the retina's compensatory hyperemic response to ischemia, hypoxia, and hypoglycemia. Increasing endogenous adenosine concentrations may be useful in ameliorating post-ischemic hypoperfusion. Overall, current evidence suggests that adenosine is a vital component of the endogenous retinal response to substrate deprivation. Additionally, in vitro studies provide strong evidence that adenosine is a mediator of the formation and effects of vascular endothelial growth factor, which in turn promotes neovascularization. Finally, the ability of the retina to develop an ischemia-tolerant state by ischemic preconditioning is an intriguing phenomenon that reveals yet another essential role for adenosine in the retina's endogenous response to ischemia. The experimental results described in this review suggest that continued investigation into the role of adenosine in the retina may lead to important clinical applications for adenosine-based therapies that could decrease the incidence of retinal damage in ischemic vasculopathies such as diabetes, glaucoma, and retinal vascular occlusion.
Assuntos
Traumatismo por Reperfusão/fisiopatologia , Vasos Retinianos , Adenosina/análise , Adenosina/metabolismo , Adenosina/fisiologia , Animais , Bovinos , Clonagem Molecular , Retinopatia Diabética/fisiopatologia , Expressão Gênica , Cobaias , Humanos , Isquemia/fisiopatologia , Precondicionamento Isquêmico , Ratos , Receptores Purinérgicos P1 , Retina/químicaRESUMO
The authors examined the involvement of platelet-activating factor (PAF) in mediating leukocyte adherence to brain postcapillary pial venules and altering blood-brain barrier (BBB) permeability during basal conditions and during reoxygenation after asphyxia in newborn piglets. Intravital epifluorescence videomicroscopy, closed cranial windows, and labeling of leukocytes with rhodamine 6G allowed us to obtain serial measurements of adherent leukocytes within postcapillary venules. Blood-brain barrier breakdown was determined by optical measures of cortical extravascular fluorescence intensity after intravenous sodium fluorescein. Superfusion of PAF over the cortex induced a dose-dependent increase in leukocyte adherence to cerebral venules and leakage of fluorescein; with 1 micromol/L PAF, the magnitude of adherence and BBB breakdown was similar to that seen during reoxygenation after 9 minutes of asphyxia. Both adherence and loss of BBB integrity resulting from either exogenous PAF or asphyxia-reoxygenation could be significantly attenuated by intravenous administration of WEB 2086, a PAF receptor antagonist. Window superfusion of superoxide dismutase with PAF attenuated PAF-induced increases in adherence and associated fluorescein leakage. These findings indicate that PAF exhibits proinflammatory effects in piglet brain and that PAF contributes to leukocyte adherence and BBB breakdown after cerebral ischemia. These PAF effects are mediated by increases in superoxide radical generation.
Assuntos
Barreira Hematoencefálica/fisiologia , Isquemia Encefálica/fisiopatologia , Endotélio Vascular/citologia , Leucócitos/citologia , Fator de Ativação de Plaquetas/fisiologia , Animais , Animais Recém-Nascidos , Asfixia Neonatal/tratamento farmacológico , Asfixia Neonatal/fisiopatologia , Permeabilidade Capilar , Adesão Celular/fisiologia , Feminino , Hemodinâmica/fisiologia , Humanos , Recém-Nascido , Masculino , Microscopia de Fluorescência , Oxigênio/uso terapêutico , Suínos , Gravação em VídeoRESUMO
Neuroprotection against cerebral ischemia can be realized if the brain is preconditioned by previous exposure to a brief period of sublethal ischemia. The present study was undertaken to test the hypothesis that nitric oxide (NO) produced from the neuronal isoform of NO synthase (NOS) serves as a necessary signal for establishing an ischemia-tolerant state in brain. A newborn rat model of hypoxic preconditioning was used, wherein exposure to sublethal hypoxia (8% oxygen) for 3 hours renders postnatal day (PND) 6 animals completely resistant to a cerebral hypoxic-ischemic insult imposed 24 hours later. Postnatal day 6 animals were treated 0.5 hour before preconditioning hypoxia with the nonselective NOS inhibitor L-nitroarginine (2 mg/kg intraperitoneally). This treatment, which resulted in a 67 to 81% inhibition of calcium-dependent constitutive NOS activity 0.5 to 3.5 hours after its administration, completely blocked preconditioning-induced protection. However, administration of the neuronal NOS inhibitor 7-nitroindazole (40 mg/kg intraperitoneally) before preconditioning hypoxia, which decreased constitutive brain NOS activity by 58 to 81%, was without effect on preconditioning-induced cerebroprotection, as was pretreatment with the inducible NOS inhibitor aminoguanidine (400 mg/kg intraperitoneally). The protective effects of preconditioning were also not blocked by treating animals with competitive [3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate; 5 mg/kg intraperitoneally] or noncompetitive (MK-801; 1 mg/kg intraperitoneally) N-methyl-D-aspartate receptor antagonists prior to preconditioning hypoxia. These findings indicate that NO production and activity are critical to the induction of ischemic tolerance in this model. However, the results argue against the involvement of the neuronal NOS isoform, activated secondary to a hypoxia-induced stimulation of N-methyl-D-aspartate receptors, and against the involvement of the inducible NOS isoform, but rather suggest that NO produced by the endothelial NOS isoform is required to mediate this profound protective effect.
Assuntos
Isquemia Encefálica/prevenção & controle , Hipóxia/fisiopatologia , Óxido Nítrico/fisiologia , Animais , Animais Recém-Nascidos , Cálcio/farmacologia , Maleato de Dizocilpina/farmacologia , Inibidores Enzimáticos/farmacologia , Guanidinas/farmacologia , Indazóis/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/fisiologia , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Nitroarginina/farmacologia , Oxigênio/administração & dosagem , Piperazinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/fisiologiaRESUMO
Blood-brain barrier breakdown and edema, indicative of cerebrovascular injury, are characteristic pathophysiologic outcomes following head trauma. These injuries result from both primary mechanical damage and from secondary events initiated by the traumatic insult. Free radicals are recognized as mediators of secondary injury in a number of trauma models. In this study, we used a novel in vitro model of traumatic microvascular injury to test the hypothesis that endogenous glutathione protects cerebral endothelial cells from secondary autooxidative injury following mechanical trauma. Porcine brain cerebral endothelial cells were grown in tissue culture wells with Silastic membrane bottoms, and cellular injury was induced by displacing the membrane different distances with user-defined pressure pulses from a customized device. The resultant endothelial cell injury 2 h following stretch was determined by measuring lactate dehydrogenase in the culture media. Significant stretch-dependent increases in endothelial injury were elicited that depended in a nonlinear fashion on the degree of membrane displacement. Depletion of intracellular glutathione with buthionine sulfoximine (1 mM) increased the extent of traumatic endothelial cell injury by 17-56%, particularly at low to moderate levels of traumatic injury (30-40% of total endothelial cell LDH release). Conversely, traumatic injury was reduced by 22-45% when endothelial cell glutathione levels were augmented threefold (to 140+/-8 nmol/mg protein) by preincubating cells with 2 mM glutathione; the extent of protection was inversely proportional to the extent of the traumatic stretch. Traumatic endothelial cell injury was also significantly and dose-dependently attenuated (up to 40%) by treatment with the xanthine oxidase inhibitor oxypurinol (50 and 100 microM). These results demonstrate that cerebral endothelial cells are the targets of hydrogen peroxide-mediated injury secondary to trauma-induced superoxide radical formation via the xanthine oxidase pathway. The neutralization of peroxides by the endogenous glutathione redox cycle provides endothelial cells a finite capacity to reduce free radical-mediated traumatic injury; this cycle may be amenable to therapeutic manipulation to mitigate posttraumatic edema and other manifestations of vascular dysfunction.
Assuntos
Encéfalo/irrigação sanguínea , Endotélio Vascular/lesões , Glutationa/farmacologia , Animais , Animais Recém-Nascidos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Butionina Sulfoximina/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , Radicais Livres/metabolismo , Glutationa/metabolismo , Glutationa/fisiologia , Técnicas In Vitro , Líquido Intracelular/metabolismo , L-Lactato Desidrogenase/metabolismo , Oxipurinol/farmacologia , Suínos , Xantina Oxidase/antagonistas & inibidoresRESUMO
Although reductions in retinal blood flow (RBF) in response to acute hyperoxia are well described, the mechanistic basis of this response has yet to be clarified. The present study was undertaken in order to determine the possible involvement of two arachidonic acid-derived vasoconstrictors, the cyclooxygenase metabolite thromboxane and the cytochrome P450 metabolite 20-HETE, as well as the involvement of the peptide endothelin and superoxide free radical. Fluorescein videoangiography was performed on the intact eyes of isoflurane-anesthetized newborn piglets. RBF responses to 20 min of hyperoxia were calculated from the angiograms off-line, using changes in mean arteriovenous transit times and arteriolar and venular diameters. The effect of hyperoxia (PaO2=351+/-9 mmHg; n=39) on RBF was examined in each animal under control conditions and again after intravitreal perivascular administration of drugs that block the synthesis or receptors of known vasoconstrictors. Estimated RBF decreased by a maximum of 42+/-3% in the 7 animal groups in response to 20 min of hyperoxia. The magnitude and time course of the change in RBF resulting from two successive hyperoxic challenges did not differ, and were unaffected by intravitreal administration of vehicle. The response to hyperoxia was attenuated 46+/-6 (n=6; P=0.001) after intravitreal CGS 22652 (2 nmol), a combined thromboxane synthesis inhibitor and receptor antagonist. DDMS (12.5 nmol), a competitive inhibitor of the P450 enzyme omega-hydroxylase that forms 20-HETE, blocked hyperoxic constriction by 23+/-7% (n=6; P=0.01). Intravitreal pretreatment with TBC 1241z (2 nmol), a receptor antagonist of the peptide endothelin, blocked the hyperoxic response by 26+/-5% (n=6; P=0.01). A combination of CGS 22652 (2 nmol), DDMS (12.5 nmol), and TBC 1241z (2 nmol), blocked the hyperoxic flow response by 51+/-3% (n=5; P=0.003). Administration of a combination of superoxide dismutase (10 U intravitreally, 10000 U kg-1 of the polyethylene glycol-conjugate intravenously) and catalase (10 U intravitreally, 10000 U kg-1 intravenously) was without effect on hyperoxia-induced reductions in RBF (n=5). The present results indicate that the arachidonic acid metabolites thromboxane and 20-HETE, and the peptide endothelin, participate in mediating the acute reduction in RBF in response to hyperoxia.
Assuntos
Hiperóxia/fisiopatologia , Vasos Retinianos/fisiologia , Análise de Variância , Animais , Animais Recém-Nascidos , Caprilatos/farmacologia , Antagonistas dos Receptores de Endotelina , Inibidores Enzimáticos/farmacologia , Angiofluoresceinografia , Injeções , Receptores de Tromboxanos/antagonistas & inibidores , Fluxo Sanguíneo Regional/efeitos dos fármacos , Vasos Retinianos/efeitos dos fármacos , Sulfonamidas/farmacologia , Suínos , Tromboxanos/antagonistas & inibidores , Gravação em VídeoRESUMO
PURPOSE: Little is known regarding the status and implications of altered retinal blood flow (RBF) following a period of temporary retinal ischemia. We undertook studies to measure acute changes in RBF after ischemia, and the mechanisms responsible for mediating these changes. METHODS: Retinal ischemia was induced in anesthetized, mechanically ventilated newborn pigs by severe hypoxia, hypotension, and bradycardia secondary to 9 min of asphyxia by discontinued ventilation. Using fluorescein videoangiography, we calculated stimulus-induced changes in RBF by measuring changes in arteriovenous transit times and arteriolar and venular diameters from the angiogram videorecordings. RESULTS: Asphyxia led to a progressive reduction in RBF during early reperfusion, with RBF decreasing 24 +/- 6% and 34 +/- 5% below baseline 1 h and 2 h, respectively, after asphyxia (n = 6). Intravitreal administration of the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (25 nmol) at 15 min of postischemic reperfusion did not increase the magnitude of hypoperfusion (n = 6), and intravitreal acetylcholine (20 nmol) was no longer able to increase RBF at 1.5-2.0 h of postasphyxic reperfusion. The endothelin A receptor antagonist TBC 11251z attenuated the response by 53% at 2 h (n = 5). The adenosine transport inhibitor 4-nitrobenzyl-6-thioinosine reversed the hypoperfusion response (n = 5), whereas ventilating animals with 100% oxygen during reperfusion exacerbated the flow deficit, with RBF reduced to 49 +/- 5% below baseline at 2 h post-asphyxia (n = 6). CONCLUSIONS: These findings indicate that (1) constriction by endothelin, together with a loss of nitric oxide's tonic dilatative effect, contributes importantly to mediating postischemic hypoperfusion in retina; (2) improvements in retinal perfusion can be realized with endothelin receptor blockade or potentiation of extracellular adenosine; and (3) additional reductions in postischemic RBF can occur in response to resuscitation with 100% oxygen because retinal microcirculatory reactivity to hyperoxia remains intact during the hypoperfusion period.
Assuntos
Endotélio Vascular/fisiopatologia , Isquemia/fisiopatologia , Vasos Retinianos/fisiopatologia , Acetilcolina/farmacologia , Adenosina/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Velocidade do Fluxo Sanguíneo , Antagonistas dos Receptores de Endotelina , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Angiofluoresceinografia , Hiperóxia/fisiopatologia , Hipóxia/complicações , Hipóxia/fisiopatologia , Isquemia/etiologia , Isoxazóis/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Reperfusão , Vasos Retinianos/efeitos dos fármacos , Suínos , Tioinosina/análogos & derivados , Tioinosina/farmacologia , Tiofenos/farmacologia , ômega-N-Metilarginina/farmacologiaRESUMO
BACKGROUND AND PURPOSE: Recent studies indicate that leukocytes are important contributors to secondary vascular and parenchymal injury after cerebral ischemia. The present study was undertaken to define nitric oxide (NO)-based mechanisms that regulate leukocyte-endothelial interactions in the cerebral vasculature, how these mechanisms are affected by cerebral ischemia, and whether NO-based therapies can affect postischemic leukocyte dynamics. METHODS: Leukocyte adherence to pial venules of anesthetized newborn piglets was quantified by in situ fluorescence videomicroscopy through closed cranial windows during basal conditions and during reperfusion after 9 minutes of asphyxia. Nitric oxide synthase (NOS) was inhibited by local window superfusion of L-nitroarginine; superfusion of sodium nitroprusside was used to donate NO. RESULTS: Local inhibition of NOS under resting conditions increased leukocyte-endothelial adherence 2.2-fold and 3.9-fold over baseline values after 1 hour and 2 hours, respectively; this response was completely blocked by cosuperfusion with L-arginine. Cosuperfusion of superoxide dismutase reversed L-nitroarginine-induced leukocyte adherence by 89% and 63% at these respective time points. The extent of acute leukocyte adherence elicited by NOS inhibition was similar in magnitude to that observed during the initial 2 hours of reperfusion after asphyxia. Leukocyte adherence was not additionally increased in asphyxic animals treated with L-nitroarginine. Sodium nitroprusside robustly inhibited asphyxia-induced leukocyte adherence back to control levels. CONCLUSIONS: NO exerts a tonic antiadherent effect in the cerebral microcirculation by inactivation of adherence-promoting superoxide radical formation. Cerebral ischemia is associated with an inhibition of NOS or lower levels of NO, which results in leukocyte-endothelial adherence that can be prevented by NO donors. The latter may be useful therapeutically to prevent the purported vascular and parenchymal dysfunction and injury caused by activated leukocytes in ischemic brain.
Assuntos
Isquemia Encefálica/fisiopatologia , Endotélio Vascular/fisiopatologia , Leucócitos/fisiologia , Óxido Nítrico/farmacologia , Animais , Animais Recém-Nascidos , Isquemia Encefálica/patologia , Adesão Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Nitroarginina/farmacologia , Nitroprussiato/farmacologia , Pia-Máter/irrigação sanguínea , Valores de Referência , Suínos , Vasodilatadores/farmacologia , Vênulas/fisiopatologiaRESUMO
Programmed cell death (apoptosis) is a normal process in the developing nervous system. Recent data suggest that certain features seen in the process of programmed cell death may be favored in the developing versus the adult brain in response to different brain injuries. In a well characterized model of neonatal hypoxia-ischemia, we demonstrate marked but delayed cell death in which there is prominent DNA laddering, TUNEL-labeling, and nuclei with condensed chromatin. Caspase activation, which is required in many cases of apoptotic cell death, also followed a delayed time course after hypoxia-ischemia. Administration of boc-aspartyl(OMe)-fluoromethylketone, a pan-caspase inhibitor, was significantly neuroprotective when given by intracerebroventricular injection 3 h after cerebral hypoxia-ischemia. In addition, systemic injections of boc-aspartyl(OMe)-fluoromethylketone also given in a delayed fashion, resulted in significant neuroprotection. These findings suggest that caspase inhibitors may be able to provide benefit over a prolonged therapeutic window after hypoxic-ischemic events in the developing brain, a major contributor to static encephalopathy and cerebral palsy.
Assuntos
Clorometilcetonas de Aminoácidos/uso terapêutico , Isquemia Encefálica/tratamento farmacológico , Inibidores de Cisteína Proteinase/uso terapêutico , Hipóxia/tratamento farmacológico , Clorometilcetonas de Aminoácidos/administração & dosagem , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Artérias Carótidas/cirurgia , Cumarínicos/metabolismo , Inibidores de Cisteína Proteinase/administração & dosagem , Dano ao DNA/efeitos dos fármacos , Injeções Intraperitoneais , Injeções Intraventriculares , Ligadura , Oligopeptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Coloração e Rotulagem/métodos , Fatores de TempoRESUMO
Oxygen free radicals, generated by cerebral ischemia, have been widely implicated in the damage of vascular endothelium. Endothelial cells have been proposed as a significant source of oxygen free radicals. In the present study, we developed an anoxia-reoxygenation (AX/RO) model using pure cultures of cerebral endothelial cells (CECs) isolated from piglet cortex to measure CEC oxygen free radical production and determine its role in AX/RO-induced CEC injury. CEC injury, as measured by lactate dehydrogenase efflux into the culture medium, increased progressively with the duration of anoxic exposure, becoming significant after 10 h. Reoxygenation significantly increased CEC anoxic injury in a time-dependent manner. A 55% increase in oxygen free radical production, determined by fluorescence detection of dihydroethidium oxidation, was measured at the end of 4-h reoxygenation in CECs subjected to AX/RO conditions that killed 40% of the cells. Blockade of oxygen free radical production with superoxide dismutase (SOD; 250 and 1000 U/ml) or oxypurinol (50 and 200 microM), a potent xanthine oxidase inhibitor, reduced this injury by 32-36% and 30-39%, respectively. Results from our in vitro model indicate that CECs produce significant amounts of oxygen free radicals following ischemia, primarily from the xanthine oxidase pathway. These radicals ultimately have a cytotoxic effect on the very cells that produced them. Thus, reductions in oxygen free radical-mediated vascular injury may contribute to improvements in neurophysiologic outcome following treatment with oxygen free radical inhibitors and scavengers.
Assuntos
Isquemia Encefálica/fisiopatologia , Circulação Cerebrovascular/fisiologia , Endotélio Vascular/fisiopatologia , Traumatismo por Reperfusão/fisiopatologia , Superóxidos/metabolismo , Xantina Oxidase/metabolismo , Animais , Isquemia Encefálica/patologia , Células Cultivadas , Endotélio Vascular/patologia , Inibidores Enzimáticos/farmacologia , Microcirculação/fisiologia , Oxipurinol/farmacologia , Traumatismo por Reperfusão/patologia , Superóxido Dismutase/metabolismo , Superóxidos/antagonistas & inibidores , Suínos , Xantina Oxidase/antagonistas & inibidoresRESUMO
PURPOSE: The objectives of this study were to examine whether preconditioning can decrease ischemic damage to the retina, by electroretinographic assessment of visual function and by histologic examination of retinal structure; to investigate the time course of the effectiveness of preconditioning; and to determine whether protein synthesis is involved. METHODS: Retinal ischemia was produced for 60 minutes in anesthetized Sprague-Dawley rats. Recovery after ischemia was measured by electroretinography for a maximum period of 7 days. Retinal sections that were sliced 1 micron thick were examined 7 days after ischemia. Retinal ischemia for 5 minutes constituted the preconditioning stimulus. To assess the time course of preconditioning, animals first underwent preconditioning and then 60 minutes of ischemia 1, 24, 72, or 168 hours later; or they underwent a 5-minute sham experiment and 60 minutes of ischemia 24 hours later. An additional group of rats received 0.4 mg/kg cycloheximide, the protein synthesis inhibitor, intraperitoneally before preconditioning and underwent 60 minutes of ischemia 24 hours later. RESULTS: In contrast to the nonpreconditioned rats, preconditioned rats had complete recovery of the a- and b-waves compared with preischemic baseline amplitudes, and ischemia-induced histologic damage was completely prevented when preconditioning was performed 24 or 72 hours (but not 168 hours) before ischemia. Separation of preconditioning and 60 minutes of ischemia by 1 hour caused an even greater impairment of functional retinal recovery compared with that seen in sham-preconditioned rats. Severe histologic damage was also noted. Block of protein synthesis by cycloheximide completely attenuated the protective effect of preconditioning. CONCLUSIONS: Preconditioning induces profound retinal tolerance to ischemia in vivo. The absence of a protective effect of preconditioning when there was a 1-hour or a 168-hour separation between the preconditioning stimulus and ischemia and the inhibition of preconditioning by cycloheximide support the hypothesis that a transient change in protein expression is necessary to provide this protection.
Assuntos
Precondicionamento Isquêmico , Traumatismo por Reperfusão/prevenção & controle , Vasos Retinianos/fisiopatologia , Animais , Cicloeximida/farmacologia , Eletrorretinografia , Proteínas do Olho/fisiologia , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Retina/fisiologia , Doenças Retinianas/patologia , Doenças Retinianas/fisiopatologia , Doenças Retinianas/prevenção & controleRESUMO
The purpose of this study was to compare, in rats, brain microdialysis results obtained using microdialysis probes implanted acutely for 2 h versus probes implanted chronically for 24 h in the caudate. Specific comparisons included: (1) dialysate purine and amino acid profiles during cerebral ischemia; (2) diffusional characteristics of the microdialysis probe; and (3) tissue morphology surrounding the probe. During ischemia, the increase in dialysate levels of adenosine, inosine, and hypoxanthine was less pronounced from probes implanted chronically, while dialysate xanthine levels increased to a greater extent. An increase in dialysate amino acid neurotransmitters during cerebral ischemia was observed in the acutely implanted probes within 10 min of the onset of cerebral ischemia; in the chronically implanted probes this increase did not occur until after 50 min of severe ischemia. Both in vitro and in vivo tests revealed a diffusional barrier in chronically implanted probes. Moreover, the tissue surrounding chronically implanted probes exhibited a high degree of inflammation, and fibrin deposits were substantial. In addition, uric acid levels (an indicator of tissue injury) sampled from chronically implanted probes were 7-fold greater than levels sampled from acutely implanted probes. These data raise concerns about the use of chronically implanted microdialysis probes for the measurement of purine and amino acid profiles during cerebral ischemia.
Assuntos
Isquemia Encefálica/diagnóstico , Monitorização Fisiológica/métodos , Adenosina/farmacologia , Animais , Cromatografia Líquida de Alta Pressão , Difusão , Hemodinâmica/efeitos dos fármacos , Imuno-Histoquímica , Implantes Experimentais , Masculino , Microdiálise/instrumentação , Neurotransmissores/farmacologia , Nucleotídeos de Purina/farmacologia , Ratos , Ratos WistarRESUMO
Hypoxia increases brain adenosine concentrations, which provides neuroprotection through activation of central adenosine A1 receptors. This study was carried out to determine whether PD 81,273, which increases adenosine's binding to A1 receptors, would reduce hypoxia-induced brain injury. PD 81,273 (3 mg/kg, i.p.) decreased by about 50% the weight loss of the left cerebral hemisphere caused by hypoxia-ischemia in neonatal rats. Thus, enhancing adenosine's binding to the A1 receptor decreases hypoxic brain damage.
Assuntos
Lesões Encefálicas/metabolismo , Isquemia Encefálica/metabolismo , Hipóxia/metabolismo , Agonistas do Receptor Purinérgico P1 , Tiofenos/farmacologia , Animais , Animais Recém-Nascidos/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Recent accumulating evidence indicates that leukocytes contribute importantly to ischemic brain injury. Although large numbers of leukocytes are present in the ischemic territory of reperfused brain 24-48 h after the ischemic insult, little is known of the acute inflammatory response to cerebral ischemia, particularly regarding the time course and magnitude of leukocyte adherence to cerebrovascular endothelium and the functional consequences of such adherence. To study these issues, we developed an epifluorescence videomicroscopy system for observing and quantifying the dynamic behavior of rhodamine-labeled leukocytes in the cerebrovascular microcirculation. Anesthetized piglets equipped with closed cranial windows were used in these investigations. During the initial 2 h of reperfusion after 9 min of asphyxia (n = 6), a marked, progressive increase in adherent leukocytes was noted in cerebral postcapillary venules that was significantly greater in magnitude than that seen in nonasphyxic, time-matched controls (n = 8). A similar response was observed after complete global ischemia of 10 min duration. A significant increase in sodium fluorescein permeability was also measured at 2 h of reperfusion in asphyxic animals. Pretreating a separate asphyxic animal group (n = 7) with a monoclonal antibody to the leukocyte adhesion glycoprotein complex CD11/CD18 severely attenuated both leukocyte adherence and the increase in vascular permeability. These results provide evidence that adherent leukocytes contribute to disruption of endothelial integrity during early reperfusion after global ischemic insults, the inhibition of which may reduce the vasogenic edema that occurs early during reperfusion after birth asphyxia, stroke, and cardiac arrest.
Assuntos
Isquemia Encefálica/fisiopatologia , Antígenos CD18/fisiologia , Leucócitos/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos CD18/imunologia , Permeabilidade Capilar , Adesão Celular/fisiologia , Circulação Cerebrovascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Feminino , Hemodinâmica , Masculino , SuínosRESUMO
Hypoxic-ischemic brain injury in survivors of perinatal asphyxia is a frequently encountered clinical problem for which there is currently no effective therapy. Neurotrophins, such as brain-derived neurotrophic factor (BDNF), can protect responsive neurons against cell death in some injury paradigms. While the role of BDNF in hypoxic-ischemic brain injury is not clear, evidence suggests that BDNF may have different effects in the developing, as opposed to the adult, brain. We found that a single intracerebroventricular (ICV) injection of BDNF resulted in rapid and robust phosphorylation of trk receptors in multiple brain regions in the postnatal day (PD) 7 rat brain. BDNF also markedly protected against hypoxic-ischemic brain injury at PD7. It protected against 90% of tissue loss due to hypoxic-ischemia when given just prior to the insult and against 50% of tissue loss when give after the insult. In contrast, ICV injection of BDNF in PD21 and adult rats resulted in little trk phosphorylation and less dramatic protection against unilateral hypoxic-ischemic injury at PD21. Because of its potent neuroprotective actions in the developing brain, BDNF may be a potential treatment for asphyxia and other forms of acute injury in the perinatal period.